This invention relates generally to an apparatus and method for DNA sequencing. More particularly, it relates to an apparatus and method for DNA sequencing based on the direct observation of polymerase with a nanometer scale electrometer.
DNA sequencing is a major challenge. For instance, the process of determining the exact order of the 3 billion chemical building blocks making up the DNA of the 24 different human chromosomes has been and still is the greatest technical challenge in science and technology. The resulting DNA sequence maps will help reveal the estimated 100,000 human genes within our DNA as well as the regions controlling them that can then be used by 21st century scientists to explore human biology and other complex phenomena.
Transcription is the process by which the nucleotide sequence of DNA is replicated into RNA. Transcription starts with unwinding DNA at the beginning of a gene. Then nascent RNA is synthesized by a complementary base pairing with the template strand of DNA. The synthesis site moves along template DNA. Further reference for the transcription process can be found in a book by Paul Berg and Maxine Singer, entitled xe2x80x9cDealing with Genesxe2x80x9d, published by University Science Books, Mill Valley, 1990. This process is accomplished with a class of proteins called RNA polymerases. These proteins directly replicate the sequence of a template strand of DNA by constructing a nascent RNA strand from individual nucleotides. Transcription occurs when the RNA polymerase binds to a discrete DNA sequence that defines the beginning of a given gene. The discrete sequence, known as a promoter region, signals the RNA polymerase to separate the two strands of the DNA and begin replicating one of the strands into a strand of nascent DNA. Each nucleotide that is added to the nascent RNA is determined by complementary base pairing with successive nucleotides of the template DNA. For example, an A, G, T, or C in the DNA template strand correspond to a U,C, A or G in the nascent RNA strand. The polymerase moves along the template DNA while it continues to add nucleotides to the nascent RNA until the end of the gene is reached. At this point, the transcription is terminated and the completed strand of RNA is released.
The movement of RNA polymerase relative to the template DNA has been previously observed both by force detection and optical methods. Schafer et al. (1991) in a paper entitled xe2x80x9cTranscription of single molecules of RNA polymerase observed by light microscopyxe2x80x9d, published in Nature 352, page 444, observed transcription by a single RNA polymerase molecule using light microscopy to detect the Brownian motion of a gold particle that was attached to the template. Analysis of the Brownian motion enabled Schafer et al. to measure the movement of the template DNA relative to the polymerase molecule. Yin et al. (1995) in a paper entitled xe2x80x9cTranscription against an applied forcexe2x80x9d, published in Science 270, page 1653, demonstrated that the force produced by a single RNA polymerase could be measured with an optical trap. At the start of the transcription, the RNA polymerase is immobilized on a glass slide. One end of the template DNA is attached to a polystyrene bead in the optical trap while the other end is bound to the polymerase. During transcription, force exerted by the polymerase on the bead was monitored as a function of time by measuring the bead position with an interferometer. While both of these experiments give direct evidence that the RNA is indeed replicating, they do not detect specific nucleotide sequence.
U.S. Pat. No. 6,280,939 to Veeco Instruments, Inc. teaches a method and apparatus for DNA sequencing using a local sensitive force detector that is performed in real time using an atomic force microscope (AFM). The AFM""s probe detects motions that occur when a polymerase incorporates nucleotides into a growing polynucleotide chain and a newly formed double-stranded polynucleotide helix translocates (or xe2x80x9cratchetsxe2x80x9d) through the polymerase""s reaction site. These motions generate a mechanical force that is reflected, either directly or indirectly, by motion of the AFM cantilever. The resulting changes in cantilever motion are detected and can be recorded as an indication that a nucleotide has been incorporated into the DNA template. To determine which nucleotide type has been incorporated, a characteristic of the incorporation of at least one nucleotide type of interest is flagged so as to be distinguishable from the corresponding characteristics of the incorporation of nucleotides of other types.
U.S. Pat. No. 6,238,871 to Sequenom, Inc. teaches a method to sequence DNA by mass spectrometry. The improvements of this method over the existing DNA sequencing technologies are high speed, high throughput, no electrophoresis and gel reading artifacts due to the complete absence of an electrophoretic step, and no costly reagents involving various substitutions with stable isotopes. U.S. Pat. No. 6,238,871 utilizes the Sanger sequencing strategy and assembles the sequence information by analysis of the nested fragments obtained by base-specific chain termination via their different molecular masses using mass spectrometry, as for example, MALDI or ES mass spectrometry. A further improvement U.S. Pat. No. 6,238,871 teaches is the throughput that can be obtained by introducing mass-modifications in the oligonucleotide primer, chain-terminating nucleoside triphosphates and/or in the chain-elongating nucleoside triphosphates, as well as using integrated tag sequences which allow multiplexing by hybridization of tag specific probes with mass differentiated molecular weights.
Accordingly, the demand and importance of DNA sequencing continuously requires the emergence of new methods and techniques that allow for increased DNA sequencing speed and reliability.
The present invention provides an apparatus and method for nucleotide or DNA sequencing by monitoring the molecular charge configuration as the DNA moves through a protein, for instance a RNA polymerase, that is capable of transcribing the DNA. The apparatus and method provides a nanoscale electrometer that immobilizes the protein. The protein receives the DNA and transcribes the DNA. The nanoscale electrometer is a sensitive device that is capable of sensing and measuring the electronic charge that is released during the transcription process. The apparatus and method of the present invention further provides monitoring means that are attached to the nanoscale electrometer to monitor the electronic charge configuration as the DNA moves through the protein. Once the electronic charge configuration is established, a correlation is computed, using computing means, between the electronic charge configuration and a nucleotide signature of the DNA. The present invention provides two exemplary embodiments for nanoscale electrometers; first a single electron transistor and second a nanoparticle device. In case of the single electron transistor, the protein is immobilized on the gate of the single electron transistor to receive the DNA. In case of the nanoparticle device, which includes two electrodes and a nanoparticle positioned in between the two electrodes, the protein is immobilized on the nanoparticle to receive the DNA.
The method of the present invention for sequencing DNA provides the steps of immobilizing a protein that is capable of transcribing DNA on a nanoscale electrometer and delivering the DNA to the protein. The method further provides the step of monitoring an electronic charge configuration at the nanoscale electrometer as the DNA moves through the protein. The method also includes the step of computing, using computing means, a correlation between the electronic charge and a nucleotide signature of the DNA.
The present invention further provides an integrated circuit chip for sequencing one or more DNA samples. The integrated circuit chip includes a plurality of interconnected nanoscale electrometers and a plurality of proteins that are capable of transcribing one or more DNA samples. The proteins are immobilized on the interconnected nanoscale electrometers to receive and transcribe one or more DNA samples.
The present invention further provides a method for sequencing one or more DNA samples with the steps of immobilizing a plurality of proteins that are capable of transcribing DNA samples on a plurality of nanoscale electrometers and delivering the DNA samples to the proteins. The method further provides the step of monitoring electronic charge configurations at the nanoscale electrometers as the DNA moves through the proteins. The method also provides the step of computing one or more correlations between the electronic charge configurations and nucleotide signatures of the DNA.
In view of that which is stated above, it is an objective of the present invention to provide an apparatus and method for DNA sequencing with a transcription protein and a nanoscale electrometer. An advantage of the present invention over the prior art is that the system enables one to directly measure the DNA sequence as the transcription process unfolds.